Method for the simultaneous detection of hybridization and vaccination reactions and diagnostic uses thereof

ABSTRACT

The present invention relates to a method for simultaneously detecting hybridization reactions and immunoreactions in a sample which may contain target analytes consisting of at least one nucleic acid and of at least one other ligand that is different in nature, characterized in that it comprises the steps consisting in: (i) depositing a known amount of volume of the sample diluted in a reaction buffer, onto a capture surface pre-coated with the partners for capturing said target analytes, said capture partners consisting of at least one nucleic acid probe and at least one antiligand, (ii) reacting at a temperature of between 15° C. and 60° C. and (iii) visualizing the hybridization reactions and immunoreactions thus obtained, and also to the use of this method for detecting diseases of infectious or metabolic origin or viral origin, for the industrial diagnosis of the presence of bacteria, and for identifying and/or quantifying biological molecules. The invention also relates to the use of this method in diagnosis and the biological or diagnostic test kits that are useful for carrying out the method of the invention.

The present invention relates to a novel method for simultaneouslydetecting hybridization reactions and immunoreactions, and also to itsuse in therapeutic and industrial diagnosis and in the identificationand/or quantification of biological molecules.

The diagnosis or the monitoring of pathologies most commonly requireshybridization detections and/or immunodetections. Thus, in the case ofAIDS diagnosis, it may be necessary to investigate both the presence ofthe p24 protein, of the anti-viral envelope protein antibody and of theviral RNA, or of anti-p24 protein antibody and of the viral RNA.

The simultaneous detection of the hybridization reactions andimmunoreactions under the same operating conditions and in the samereaction medium would make it possible to facilitate the diagnosis ofpathologies requiring such a double detection.

The detection of the immunoreactions and of hybridization reactionsusing common parameters has already been studied in the prior art.

Thus, patent application WO 01/86296 describes a method designed for thesimultaneous detection of a large number of analytes using a panel ofmarkers specific for each analyte. However, this method does notdescribe the simultaneous detection both of hybridization reactions andof immunoreactions, and uses operating conditions for visualization thatare different for each marker specific for each analyte.

Patent application WO 01/61040 also describes a method forsimultaneously detecting analytes using semiconducting nanocrystals asdetection markers. The reading of the hybridization reactions orimmunoreactions is carried out by means of the variation in wavelengthfor the reading. However, the disadvantage of this method is that thesample to be analyzed must be divided up into several samples, such thateach analyte to be studied is subjected to different operatingconditions.

The methods of the prior art therefore have the drawbacks thatsimultaneous detection of hybridization reactions and of immunoreactionscannot take place under the same operating conditions and in the samereaction medium, such that the simultaneous detection is complex andexpensive.

The applicant has now developed a novel method for simultaneouslydetecting hybridization reactions and immunoreactions which overcomesthe abovementioned drawbacks.

Thus, a subject of the invention is a method for simultaneouslydetecting hybridization reactions and immunoreactions in a sample whichmay contain target analytes consisting of at least one nucleic acid andof at least one other ligand that is different in nature, characterizedin that it comprises the steps consisting in:

-   -   (i) depositing a known amount of volume of the sample diluted in        a reaction buffer, onto a capture surface pre-coated with the        partners for capturing said target analytes, said capture        partners consisting of at least one nucleic acid probe and at        least one antiligand,    -   (ii) reacting at a temperature of between 15° C. and 60° C. and    -   (iii) visualizing the hybridization reactions and        immunoreactions thus obtained, and also the use of this method        for detecting diseases of infectious, for example viral, and        metabolic origin, for the industrial diagnosis of the presence        of bacteria, and also for identifying and/or quantifying        biological molecules.

Another subject of the invention consists of biological or diagnostictest kits that are useful for carrying out the method of the invention.

The method of the invention is a method simple to carry out, which makesit possible, against all expectations, to detect, in a sample, under thesame operating conditions, namely the reaction medium and temperature,the presence of target analytes consisting of at least one nucleic acidand of at least one other ligand that is different in nature. Thepresence of said target analytes is demonstrated by means of thevisualization of hybridization reactions and of immunoreactions.

The term “hybridization reaction” is intended to mean any reactionbetween a capture nucleic acid and a target nucleic acid, and the term“immunoreaction” is intended to mean any reaction between a captureantiligand and a target ligand that is other than nucleic acid innature.

The term “nucleic acid” is intended to mean oligonucleotides,deoxyribonucleic acids and ribonucleic acids, and derivatives thereof.

The term “oligonucleotide” denotes a chain of at least 2 nucleotides(deoxyribonucleotides or ribonucleotides, or both), that are natural ormodified, capable of hybridizing, under suitable hybridizationconditions, with an at least partially complementary oligonucleotide.The term “modified nucleotide” is intended to mean, for example, anucleotide comprising a modified base and/or comprising a modificationin terms of the internucleotide bond and/or in terms of the backbone. Byway of example of a modified base, mention may be made of inosine,methyl-5-deoxycytidine, dimethylamino-5-deoxyuridine, diamino-2,6-purineand bromo-5-deoxyuridine. To illustrate a modified internucleotide bond,mention may be made of phosphorothioate, N-alkylphosphoramidate,alkylphosphonate and alkylphosphodiester bonds. Alpha-oligonucleotidessuch as those described in FR-A-2 607 507, LNAs such asphosphorothioate-LNA and 2′-thio-LNA described in Bioorganic & MedicinalChemistry Letters, Volume 8, Issue 16, Aug. 18, 1998, pages 2219-2222,and the PNAs which are the subject of the article by M. Egholm et al.,J. Am. Chem. Soc. (1992), 114, 1895-1897, are examples ofoligonucleotides consisting of nucleotides whose backbone is modified.

The expression “ligand that is different in nature, other than nucleicacid in nature” is intended to mean any molecule that is different froma nucleic acid, capable of binding with a specific binding partner. Thisbinding partner is called an antiligland when it consists of the capturepartner. For reasons of convenience, the terms “ligand” and “antiligand”will be used hereinafter to denote any compound capable of animmunoreaction, but not of a hybridization reaction.

As a ligand, mention may be made, for example, of antigens, antibodies,polypeptides, proteins, haptens, sugars, enzymes and their substrates.

As an antiligand, mention may be made of the same examples as thoseregarding the ligands, and also lectins, cell receptors and aptamers.

The term “antigen” denotes a compound capable of being recognized by anantibody whose synthesis it has induced by means of an immune response.

The term “antibody” includes polyclonal and monoclonal antibodies,antibodies obtained by genetic recombination and antibody fragments.

The polyclonal antibodies can be obtained by immunization of an animalwith at least one target antigen of interest, followed by recovery ofthe desired antibodies in purified form, by taking the serum of saidanimal, and separating said antibodies from the other serumconstituents, in particular by affinity chromatography on a column towhich is attached an antigen specifically recognized by the antibodies,in particular a target antigen of interest.

The monoclonal antibodies can be obtained by the hybridoma technique,the general principle of which is recalled below.

Firstly, an animal, generally a mouse (or cells in culture in the caseof in vitro immunizations), is immunized with a target antigen ofinterest, for which the B lymphocytes are then capable of producingantibodies against said antigen. These antibody-producing lymphocytesare then fused with “immortal” myeloma cells (murine cells in theexample) so as to produce hybridomas. From the heterogeneous mixture ofthe cells thus obtained, a selection of the cells capable of producing aparticular antibody and of multiplying indefinitely is then carried out.Each hybridoma is multiplied in the form of a clone, each one resultingin the production of a monoclonal antibody whose properties ofrecognition with respect to the tumor antigen of interest may be tested,for example by ELISA, by one- or two-dimensional immunoblocking, byimmunofluorescence, or using a biosensor. The monoclonal antibodies thusselected are subsequently purified, in particular according to theaffinity chromatography technique described above.

The antibody fragments are such that they conserve the function ofbinding with their binding or capture partner.

The term “polypeptide” is intended to mean a chain of at least two aminoacids. The term “amino acids” is intended to mean the primary aminoacids which encode proteins, the amino acids derived after enzymaticaction such as trans-4-hydroxyproline and the amino acids that arenatural but not present in proteins, such as norvaline,N-methyl-L-leucine, staline (Hunt S. in Chemistry and Biochemistry ofthe amino acids, Barett G C, ed., Chapman and Hall, London, 1985), theamino acids protected with chemical functions that can be used insolid-support synthesis or in liquid phase, and the unnatural aminoacids.

The term “protein” includes holoproteins and heteroproteins, such asnucleoproteins, lipoproteins, phosphoproteins, metalloproteins andglycoproteins, both fibrous and globular.

The term “hapten” denotes nonimmunogenic compounds, i.e. compoundsincapable by themselves of promoting an immune reaction by antibodyproduction, but capable of being recognized by antibodies obtained byimmunization of animals under known conditions, in particular byimmunization with a hapten-protein conjugate. These compounds generallyhave a molecular mass of less than 3000 Da, and most commonly less than2000 Da, and may be, for example, glycosylated peptides, metabolites,vitamins, hormones, prostaglandins, toxins or various medicinalproducts, nucleosides and nucleotides.

The enzymes and their substrates are well known to those skilled in theart. The use of an enzyme as a capture antiligand makes it possible tosearch for substrate analogs for the enzyme, in the sample tested.

The lectins are capable of recognizing sugars by a mechanism well knownto those skilled in the art, as described in Biochemistry, 4th Edition,G. L. Zubay, 1998, The Mc Graw-Hill Companies, USA, Boston.

The receptors used as antiligands are surface proteins capable ofbinding with the envelope protein target ligand. Examples of suchreceptors include the CXCR4 chemokine receptor for the HIV gp120envelope protein (Corananiti, M. T., 2001, Neuroscience Letters, 312(2),67-70).

The aptamers are capture partners that are protein and nucleic acid innature, the function of which is to act as antibodies and to bind toprotein ligands (Toulmé, J. J. and Giege, R., 1998, Medecine Science,14(2), 155-166).

The sample tested in the method of the invention may be eitherbiological or industrial.

As a biological sample, mention may be made of any biological fluidcapable of containing target analytes such as blood, lymph,cerebrospinal fluid, a throat swab, vaginal smears, and urine.

As an industrial sample, mention may be made of any sample originatingfrom industry, for which a biological analysis is necessary. Samplesoriginating from the food industry (ready-prepared meals, treated water)constitute an example of an industrial sample.

The reaction buffer in which the sample is diluted is an intermediatereaction buffer between a hybridization buffer and a buffer used inimmunoreactions. It can be readily determined by those skilled in theart.

According to a particular embodiment, the reaction buffer has an ionicstrength of between 0.4 and 1 M, has a pH of between 7 and 8, andcontains a surfactant.

As a reaction buffer, mention may be made of buffers based on phosphatesalts, sodium salts, lithium salts and HEPES, and as a surfactant,mention may be made of Tween 20, Tween 80 and Triton.

The originality of the invention therefore consists in the fact that itis possible to detect both hybridization reactions and immunoreactionsin this reaction buffer.

The reaction temperature of the method of the invention is between 15and 60° C. At a temperature below 15° C., the hybridization reactionsrisk being relatively nonspecific, in particular if the oligonucleotidesare more than 8 bp in length, and at a temperature above 60° C.,problems may be encountered in terms of the ligands and antiligands.Indeed, at these temperatures, some proteins are denatured and thehybridization reactions are only possible with large oligonucleotides,longer than 30 bp.

According to one embodiment, the reaction temperature is between 35 and45° C., preferably between 37 and 41° C., the temperatures of 37 and 41°C. being more preferred.

The capture surface on which the sample to be tested is deposited can beany surface to which it is possible to attach nucleic acid probes andantiligands. As a capture surface, mention may be made of microwells,microplates, polymer surfaces, membranes, microscope slides,functionalized or unfunctionalized inorganic supports such as silica,glass, mica or quartz, metal surfaces such as gold and silver, andparticles and microparticles, in particular magnetic particles andmicroparticles.

For the method of the invention, the capture surface is precoated withthe partners for capture of said target analytes, namely nucleic acidprobes and antiligands.

The capture partners can be deposited according to the method describedin the applicant's patent application FR 00/14691 (FR 2 816 711), whichis a method of deposition without contact that is particularly suitablefor the 96-well microplate format. This method consists in ejectingcalibrated nanodroplets through a nozzle, under the effect of amechanical impact, onto the bottom of the wells of the microplate. Ateach impact, a drop whose diameter varies according to that of thenozzle, from 50 to 500 μm, approximately, is thus obtained.

The deposition of these partners can also be carried out manually usingglass capillaries having an internal diameter in the region of about ahundred microns. In this case, the capillary must come into contact withthe deposition surface in order for the deposition to take place, whichresults in a slight impact with the deposition surface.

The visualization of the hybridization reactions and immunoreactions canbe carried out by any detection means, such as direct or indirect means.

In the case of direct detection, i.e. without the use of labeling, thehybridization reactions and immunoreactions are observed, for example,by plasmon resonance or by cyclic voltametry on an electrode bearing aconducting polymer.

In the case of indirect detection, i.e. via labeling, the labeling canbe carried out either directly on the target analytes or via a bindingpartner specific for said target analytes, that is prelabeled.

The expression “binding partner specific for the target analytes” isintended to mean any partner capable of binding with the target analyte,and by way of examples, mention will be made of nucleic acids, antigens,antibodies, antibody fractions, proteins, haptens, oligonucleotides orpolynucleotides, and enzyme substrates.

According to an embodiment in which the visualization of thehybridization reactions and immunoreactions is carried out by means ofprelabeled binding partners specific for said target analytes, themethod of the invention comprises the following additional step (i′),placed between step (i) and step (ii), consisting in:

-   -   (i′) adding binding partners specific for said target analytes,        which partners have been conjugated to labels beforehand.

According to another embodiment in which the visualization of thehybridization reactions and immunoreactions is also carried out by meansof prelabeled binding partners specific for said target analytes, themethod of the invention comprises the following two additional steps(ii′) and (ii″), placed between step (ii) and step (iii), consisting in:

-   -   (ii′) adding binding partners specific for said target analytes,        which partners have been conjugated to labels beforehand, and    -   (ii″) reacting at a temperature of between 15° C. and 60° C.

Thus, in this case, the method of the invention comprises two reactionsteps, one for the binding between the partners for capturing the targetanalytes and the target analytes (step (ii)), and the other between thecapture partner/target analyte conjugate and the binding partnerspecific for the target analyte (step (ii″)).

According to yet another embodiment, the visualization of thehybridization reactions is carried out by means of the prelabeling ofthe target analytes of the nucleic acid type, and the visualization ofthe immunoreactions is carried out by means of the labeling of thebinding partners specific for said target analytes of the ligand type.Consequently, in this embodiment, since the target nucleic acids havebeen prelabeled, the only binding partners to be added in step (i′) or(ii′) are the partner(s) specific for the ligand, which has(have) beenprelabeled.

The immunoreactions can also be detected according to a “competition”method. The ligand(s) which may be contained in the biological sample,which ligand(s) has(have) been prelabeled, is(are) then added to thereaction medium (steps (i′) and (ii′)), in place of the ligand-specificpartner(s), which constitutes another embodiment of the invention. Inthis case, the detection signal is at a maximum in the absence of theligand being sought, and then gradually decreases as the concentrationof ligand being sought, which is not labeled, increases via thecompetition reaction.

The capture surface may be rinsed after the reaction steps (steps (ii)and/or (ii″)) of each embodiment of the invention, in order to removefrom the capture surface the molecules which have not reacted and themolecules that are weakly adsorbed, without specific interaction. Thus,the visualization of the hybridization reactions and immunoreactions areaccordingly improved. This constitutes another embodiment of theinvention.

As rinsing medium, use may be made, for example, of PBS-tween, asdescribed by A. Perrin et al., in J. Immunological Methods, 1999, 224,77-87.

The term “labeling” is intended to mean the attachment of a labelcapable of directly or indirectly generating a detectable signal. Anonlimiting list of these labels consists of:

-   -   enzymes which produce a signal that is detectable, for example,        by colorimetry, fluorescence or luminescence, such as        horseradish peroxidase, alkaline phosphatase, α-galactosidase or        glucose-6-phosphate dehydrogenase,    -   chromophors such as luminescent or dye compounds,    -   radioactive molecules such as ³²P, ³⁵S or ¹²⁵I,    -   fluorescent molecules such as fluorescein, rhodomine, alexa or        phycocyanins, and    -   particles such as gold particles or magnetic latex particles, or        liposomes.

Indirect systems can also be used, such as, for example, by means ofanother ligand/antiligand couple. The ligand/antiligand couples are wellknown to those skilled in the art, and mention may be made, for example,of the following couples: biotin/streptavidin, hapten/antibody,antigen/antibody, peptide/antibody, sugar/lectin, andpolynucleotide/sequence complementary to the polynucleotide. In thiscase, it is the ligand which carries the binding agent. The antiligandmay be directly detectable by means of the labels described in theparagraph above, or may itself be detectable by means of aligand/antiligand.

These indirect detection systems can result, under certain conditions,in an amplification of the signal. This signal amplification techniqueis well known to those skilled in the art, and reference may be made tothe prior patent applications FR 98/10084 or WO-A-95/08000 by theapplicant or to the article J. Histochem. Cytochem. 45: 481-491, 1997.

The prelabeling of the target analytes of the nucleic acid type can becarried out by direct or indirect incorporation of label by means of apolymerase.

The labeling of the binding partners specific for the target analytes iswidely known to those skilled in the art and is described, for example,by Greg T. Hermanson in Bioconjugate Techniques, 1996, Academic PressInc, 525B Street, San Diego, Calif. 92101 USA.

According to the type of labeling of the conjugate used, such as forexample using an enzyme, those skilled in the art will add reagents forvisualizing the labeling.

Thus, according to one embodiment of the method of the invention, thesubstrate(s) specific for the label(s) is(are) added before step (iii)for visualizing the hybridization reactions and immunoreactions.

Such reagents are widely known to those skilled in the art and aredescribed in particular in Principles and Practice of Immunoessay,2^(nd) Edition, Edited by C. Price, D. J. Newman Stockton Press, 1997,345 Park Avenue South, New York.

The elements labeled for the purposes of the method of the invention,namely the nucleic acid-specific binding partners, the ligand-specificbinding partners, the target nucleic acids contained in the biologicalsample and the ligands which may be contained in the biological sample,can be labeled with different labels or with the same label, the secondsolution being preferred.

The hybridization reactions and immunoreactions detected by the methodof the invention may be representative of the presence of a singledisease. In this case, the capture partners applied to the capturesurface are markers for the same disease, which constitutes anembodiment of the invention.

Thus for example, in the case of the early diagnosis of AIDS, it ispossible to search for the presence both of the anti-p24 proteinantibody and of the viral RNA. The capture partners present at thecapture surface may therefore be the p24 protein, and a nucleic acidprobe capable of a hybridization reaction with the viral RNA.

The hybridization reactions and immunoreactions detected by the methodof the invention can also be representative of various diseases. In thiscase, the binding partners are markers for different diseases, whichconstitutes another embodiment of the invention. Thus, the method of theinvention can be used for screening for, using a single biologicalsample, under the same operating conditions and at the same time,several different diseases demonstrated either by means of the detectionof a nucleic acid, or by means of the detection of a ligand of anothernature. This method may, for example, be useful in analyzing bloodderived from a donation, before transfusion, in which it is desired toverify that pathogens are not present.

The diseases which can be detected by means of the method of theinvention are all diseases for which at least one target analyte andcapture partner couple capable of interaction is known.

These diseases may be of infectious, namely viral or bacterial, origin,such as AIDS or the forms of hepatitis, or else they may be metabolic,such as hyperthyroidism or diabetes.

Thus, another subject of the invention consists of the use of the methodof the invention, for detecting diseases of infectious or metabolicorigin.

Yet another subject of the invention consists of the use of the methodof the invention in the industrial diagnosis of the presence ofbacteria. Indeed, the demonstration of the presence of bacteria inindustry, in particular listeria or salmonellae in the food industry,can be carried out by detection of hybridization reactions (detection ofbacterial DNA or RNA) and immunoreactions (detection of bacterialproteins).

Another subject of the invention also consists of the use of the methodof the invention, for identifying and/or quantifying biologicalmolecules. Indeed, when many different capture partners are used,demonstrating numerous different biological molecules, the method of theinvention makes it possible to identify which molecule(s) is(are)present in the sample. Similarly, when a large number of capturepartners, but with a small number of differences in nature, is used, themethod of the invention makes it possible to quantify the biologicalmolecules present in the sample, which can result in standard profilesfor a pathology or for an environmental state being defined.

Another subject of the invention consists of the use of the method ofthe invention, for simultaneously detecting the transcriptome and theproteome of a cell or of an organism. The simultaneous detection of allthe RNA (transcriptome) and of all the proteins (proteome) of a cell orof an organism thus makes it possible to demonstrate a potentialdysfunction of overexpression of the proteins relative to the RNAspresent, and vice versa.

In order to carry out the method of the invention, kits are available,consisting of at least one of the following elements:

-   -   a capture surface,    -   partners for capturing the target analytes consisting of at        least one nucleic acid probe and at least one other antiligand        that is different in nature,    -   a reaction buffer,    -   optionally, partners specific for the target ligands, that are        prelabeled, or prelabeled ligands which may be contained in the        sample to be tested, and    -   optionally, partners specific for the target nucleic acids.

These kits, which can be described as biological test kits when thesample tested is industrial, or diagnostic test kits when the sampletested is biological, constitute another embodiment of the invention.

The present invention will be understood more fully from the followingexamples, given only by way of nonlimiting illustration, and also fromthe attached FIGS. 1 and 2, in which:

FIG. 1 represents two graphs showing, firstly, the evolution of thefluorescence demonstrating the immunoreactions between the p24protein/VEMA polymer capture partner and the anti-p24 protein antibodypresent in human sera, as a function of the dilution of the sera (FIG.1A) and showing, secondly, the evolution of the fluorescencerepresentative of the hybridization reactions, or of an absence of suchreactions, where appropriate, between the nucleic acid probe capturepartners C+ (capable of reacting with the HIV virus DNA target) and C−(incapable of reacting with the HIV virus DNA target) and the HIV virusDNA target, as a function of the dilution of the PCR product (FIG. 1B),and

FIG. 2 represents the depositing plan for a multidose detection chiphaving 17 wells each comprising the capture partners in duplicate, with:

-   -   in wells 1, 9 and 17: nucleic acid capture partners for the HIV        virus (C_(HIV)),    -   in wells 3 and 11: nucleic acid capture partners for the        hepatitis B virus HBV (C_(HBV)),    -   in wells 7 and 15: nucleic acid capture partners for the        hepatitis C virus HCV (C_(HCV)),    -   in wells 5 and 13: protein capture partners for the HIV virus        (gp160 envelope protein),    -   in wells 4 and 12: protein capture partners for the HCV virus        (core protein),    -   in wells 8 and 16: protein capture partners for the HBV virus (2        different HBAg surface antigens per well), and    -   in wells 2, 6, 10 and 14: protein capture partners not specific        for this study, namely an anti-TSH antibody (NSP1, wells 2        and 10) and an anti-HCG antibody (NSP2, wells 6 and 14).

EXAMPLE 1 Preparation of the Capture Surfaces for the Detection ofHybridization Reactions and Immunoreactions for Demonstrating thePresence of the HIV Virus in a Patient

1.1 Preparation of the Capture Partner Capable of Recognizing Anti-p24Protein Antibodies

A solution of maleic anhydride vinyl ether polymer MAVE-67 (67 000g/mol) at 1 g/l in a 90/10 (V/V) mixture of DMSO (dimethylsulfoxide)/water was prepared in a flask. The mixture was left toincubate for 48 h at 37° C. 36 μg of recombinant p24 protein (pmR K24H,bioMérieux, Marcy l'Etoile, France) was then mixed with 100 μl of thepolymer solution. The mixture was left to incubate for 3 h at 37° C.

1.2 Preparation of the Capture Partner Capable of Recognizing the DNATarget Amplified from the HIV Virus RNA

Each of the following two oligonucleotides, C+ capable of hybridizationwith the DNA target and C− incapable of such a hybridization, wasdiluted to a concentration of 10 μM in 3×PBS buffer (0.45 M sodiumchloride, 0.15 M sodium phosphate, pH 6.8)-EDTA 10 mM. C+: NH₂-CGC TTCGAC AGC GAC GTG GGG C−: NH₂-TAT GAA ACT TAT GGG GAT AC

1.3 Preparation of the Capture Surfaces

4 spots were deposited without contact, according to the methoddescribed in patent application FR 00/14691 as recalled above, in eachwell of a microtitration plate (Nunc Maxisorb). Two spots consisted ofthe p24 protein-MAVE polymer conjugates, one of the spots consisted ofthe C+ probe and one consisted of the C− probe.

After deposition of the spots, the plate was immediately placed in achamber at 4° C. for 2 h, and was then incubated for 30 min at 60° C.

EXAMPLE 2 Preparation and Specific Capture of the Target Analytes

2.1 Preparation of the Target Analytes

The DNA targets (200 bp) were produced by RT-PCR from part of a viralgene extracted from cell culture. They were biotinylated using theprimers A and B below, labeled with biotin in the 5′ position: Primer A:CAT gTg CTA CTT CAC CAA Cgg Primer B: CTg gTA gTT gTg TCT gCA CAA

These targets were denatured just before their introduction, byincubation with the same volume of 0.2N sodium hydroxide for 5 min atambient temperature.

Various human sera, for which the presence or absence of anti-p24protein antibodies have been tested beforehand by conventional methods,were diluted to 1/500 in a reaction buffer A, having a pH of 7,consisting of: 0.1 M sodium phosphate, 0.5 M sodium chloride, 0.65%tween 20, 0.014% salmon DNA and 2% PEG 4000.

2.2 Specific Capture of the Target Analytes

30 μl of diluted patient serum, 30 μl of reaction buffer A and 3 μl ofdenatured DNA target were mixed and this mixture was deposited in eachwell. It was left to react for 1 h at 37° C. The wells were then rinsedwith a 50 mM sodium phosphate buffer containing 0.15 M sodium chlorideand 0.05% tween 20 (PBS-tween).

EXAMPLE 3 Detection of the Hybridization Reactions and Immunoreactions

The following mixture was deposited in each well: 30 μl of reactionbuffer A, 10 ng of a peroxidase-conjugated goat anti-human antibody(Jackson ImmunoResearch), 0.07 pmol of an oligonucleotide complementaryto the ADN target, thus making it possible to sandwich said targetbetween the capture probe immobilized at the bottom of the plate and thedetection probe. This oligonucleotide was prelabeled in the 5′ positionwith peroxidase. The reaction was left to occur for 1 h at 37° C. Thewells were then rinsed with PBS-tween.

30 μl of a colorimetric precipitating substrate for peroxidase (TMB,bioFX) were then added to each well. The visualization takes place for20 minutes. The substrate was then removed by pipetting and the opticaldensity of each of the spots was measured using a microscope (ZeissAxioplan2) connected to a CCD camera (Spot) and equipped with imageprocessing software (Image Pro+, Soft Imaging). The software makes itpossible to translate the signal intensity of each spot into a digitalvalue of between 0 (black, no signal) and 255 (white, saturatingsignal).

By way of comparison, the procedures described above were repeated, butthe DNA target was omitted in order to test the specificity ofhybridization, or sera devoid of virus (HIV−) were used.

The results obtained are given in table 1 below. TABLE 1 IntensityPresence of the of the Intensity of the Intensity of the Serum DNAtarget spot C+ spot C− spot P24-MAVE HIV+ Yes 103 48 115 HIV− Yes 108 4545 HIV+ No 47 45 109 HIV− No 46 51 48

The results above show that the specificity of the reaction is entirelysatisfactory. The presence of DNA targets does not impair therecognition immunoreaction between P24 antigen and anti-P24 antibody. Onthe contrary, hybridization of the DNA targets on the nucleic acid probespots is possible in the presence of antibodies in the medium. Thepresence of one or other of the analytes does not, either, generate anundesirable background noise. The C− spot is never detected, likewisefor the HIV− sera.

EXAMPLE 4 Quantitative and Qualitative Determination of the DetectionAccording to the Method of the Invention

The procedure described in Examples 1 and 2 was repeated, except thatthe concentration of DNA target and the dilution of the HIV+ serum werevaried.

The detection of the hybridization reactions and immunoreactions wasthen carried out as follows:

The following mixture was deposited in each well: 30 μl of reactionbuffer A, 10 ng of an alkaline phosphatase-conjugated goat anti-humanantibody (Jackson ImmunoResearch) and 25 ng of alkalinephosphatase-labeled streptavidin (Sigma). The mixture was left to reactat 1 h for 37° C. The wells were then rinsed with PBS-tween.

30 μl of a fluorescent substrate for alkaline phosphatase (ECF,Amersham, ready-to-use) were then added to each well. The visualizationtakes place for 5 min, and then the substrate was removed by pipettingand the fluorescence intensity of each of the spots was measured asindicated in Example 3 above.

The results showing the level of fluorescence relative to the dilutionof the serum or of the PCR product are represented in FIGS. 1A and 1B.For clarity reasons, the signals measured on the p24 protein-MAVEpolymer conjugate spots were reported in FIG. 1A and those measured onthe C+ spots (diamonds) and C− spots (squares), in the same well, werereported in FIG. 1B.

In FIG. 1A, a linear increase in the fluorescence signal is observed asa function of the concentration of serum in the well, and reaches aplateau for dilutions of less than 1/1000. It is thus possible toperform quantitative assays by means of this invention. The detectionlimit is estimated, according to a statistical method, at 0.00001, whichcorresponds to a serum dilution of 1/100 000. This high sensitivityallows early detection of the sera conversion subsequent to an HIVinfection.

In FIG. 1B, a similar profile to that observed with the cascadedilutions of serum is obtained. The fluorescence signal increases as afunction of the concentration of DNA target on the specific spot C+(diamonds). On the other hand, the signal remains stable, similar to itslevel measured in the absence of target, on the C− spot which cannot behybridized with the target (square). The detection limit is estimated at0.00007, i.e. a dilution of the PCR product of 1/14 000.

EXAMPLE 5 Influence of the Modification of the Reaction Temperature andBuffer

The procedure of example 4 was repeated, except that reaction buffer B,having a pH of 7.5, consisting of: 160 mM HEPES, 0.5 M lithium chlorideand 0.05% tween 20, was also used and the temperature was modified sothat it was equal to 41° C.

In this experiment, the serum contained the virus (HIV+) and the targetnot recognizing the viral DNA (C−) was absent.

The results are given in table 2 below. TABLE 2 Signal of the Signal ofthe Temperature Reaction buffer C+ spot P24-MAVE spot 37° C. A 2.1 3.837° C. B 1.8 2.7 41° C. A 1.6 3.6 41° C. B 1.3 1.8

EXAMPLE 6 Simultaneous Detection of Viral Infections Caused by the HIV,HBV and HCV Viruses in a Detection Chip

6.1 Biological Tools for Assaying Combined Nucleic Acid and ProteinParameters

6.1.1 Hybridization Reactions

HIV RNA targets obtained from Ambion (Austin, Tex., USA) were used. Thebiotinylated primers (SK431 TGCTATGTCAGTTCCCCTTGGTTCTCT and SK462AGTTGGAGGACATCAAGCAGCCATGCAAAT) and the aminated capture probes forcapturing the PCR products (C_(HIV): GAGACCATCAATGAGGAAGCTGCAGAATGGGAT)were synthesized by Eurogentec (Seraing, Belgium).

The HCV virus RNAs were extracted from sera of chronic patients usingthe Nucleospin RNA Virus Kit (Macherey-Nagel, Hoerdt, France) andamplified by RT-PCR with biotinylated primers (RC21:CTCCCGGGGCACTCGCAAGC and RC1: GTGTAGCCATGGCGTTAGTA) (Roque Afonso A. M.,2000, Journal of Virological Methods, 86, 55-60). The sequence of theaminated HCV capture probe is C_(HCV) (CATAGTGGTCTGCGGAACCGGTGAGT). TheHIV and HCV RNAs were amplified by RT-PCR under the following conditionsusing the Access kit from Promega (Madison, Wis., USA): 1×AMV/Tflreaction buffer, 1.8 mM MgSO₄, 0.2 mM dNTP, 1 μM of primers, 1USI of AMVreverse transcriptase and 5USI of Tfl DNA polymerase; cycle RT 48° C.,45 min; 35 PCR cycles (94° C., 30 s; 60° C., 1 min; 68° C., 2 min);final extension at 68° C. for 7 min.

The amplicons were analyzed on an agarose+ethidium bromide gel. Theconcentrations of the amplified products were evaluated using a massladder (Eurogentec) (46 nM for the HIV amplicons, 23 nM for the HCVamplicons).

A single-stranded synthetic target derived from the HBV gene (74 bp)(CCCAGTAAAGTTCCCCACCTTATGAGTCCAAGGAATTACTAACATTGAGATTCCCGAGATTGAGATCTTCTGCGA), an aminated capture probe (C_(HBV):ATCTCGGGAATCTCAATGTFAG) and a biotinylated detection probe (D_(HBV):TATTCCGACTCATAAGGTG), both complementary to the HBV target, weresynthesized at Eurogentec.

6.1.2 Immunoreactions

The HCV core protein (HCV core) and also the HIV envelope protein(gp160) were produced by the applicant. Two different HBV surfaceantigens (HBAgs) were used, namely a subtype Ay antigen (Hytest, Turku,Finland) and a Cliniqa plasma subtype Ad antigen (Fallbrook, Calif.,USA).

Two mouse antibodies nonspecific for this study were used, namely ananti-TSH antibody (NSP₁) and an anti-HCG antibody (NSP₂), so as to besure of the specificity of the assays.

The HIV, HBV and HCV human sera were provided by the hospitals of Lyon.

The alkaline phosphatase-labeled anti-human goat IgG conjugates wereprovided by Jackson Immunoresearch (West Grove, Pa., USA) and thealkaline phosphatase-labeled streptavidin comes from Sigma (St Quentin,France).

6.2 Preparation of the Detection Chip and Biological Assay Protocol

6.2.1 Deposition of the Capture Partners

The oligonucleotides were diluted to 10 μM in a reaction buffer foradsorption of the oligonucleotides (150 mM phosphate, 450 mM NaCl, 1 mMEDTA; pH 7.4).

The nonspecific IgGs were diluted to 50 μg/ml in a 50 mM carbonatebuffer, pH 9.3.

The gp160, HBAg and HCV core proteins were diluted to 10 μg/ml in PBS.

The deposits were made using the Biochip Arrayer device (Perkin Elmer,Boston, USA). 16+1 spots were deposited in the form of a spot, induplicate, on a circular format, in white microtitration plates(Greiner, Longwood, USA) (FIG. 2).

After deposition and incubation under controlled conditions oftemperature (10° C.) and of humidity (50%), the plates were washed(PBS-tween 0.05%), dried, and stored at 4° C.

6.2.2 Capture of the Nucleic Acid Targets and of the Antibodies

The various analyte combinations below were used:

-   I: HBV targets,-   II: HIV targets-   III: HBV serum-   IV: HIV serum-   V: HBV+HIV targets-   VI: HBV+HIV+HCV targets-   VII: HBV+HIV+HCV targets+HBV serum-   VIII: HBV+HIV+HCV targets+HBV serum+HIV serum

To do this, the following were introduced into each well: 1.5 μl of HIVor HCV amplification product, denatured beforehand with 1.5 μl of 0.2Nsodium hydroxide; 3 μl of synthetic HBV DNA at 10 nM; 1 μl of humanserum diluted to 1/10. The volume was made up to 30 μl with reactionbuffer (0.1 M Na₂HPO₄/NaH₂PO₄; 0.5 M NaCl; 0.65% tween 20; 2% PEG 4000;pH 7) and the solutions were incubated for 1 h at 37° C., and were thenrinsed with a PBS-tween 0.05% mixture.

6.2.3 Detection

The plates were incubated with a 0.2 μM solution of DHBV (HBV detection)for 30 minutes at 37° C., and were then rinsed. They were then incubatedin the presence of a solution of streptavidin-alkaline phosphatase (0.5μg/ml) and of alkaline phosphatase-labeled anti-human goat antibodies(μg/ml). The plates were then washed with PBS-tween 0.05%, and aprecipitating substrate for alkaline phosphatase was then added to eachwell (BM purple, Roche, Basel, Switzerland). The plates werephotographed using a CCD camera coupled to image analysis software,which makes it possible to determine the mean level of gray associatedwith each spot.

The results are given in table 3 below. TABLE 3 Expected Obtained Levelof gray associated Analyte position position of the with each of theanalytes combination of the spots spots (a.u.) I 3, 11 3, 11 34 935 II1, 9, 17 1, 9, 17 38 144 III 8, 16 8, 16 33 664 IV 5, 13 5, 13 12 480 V3, 11 3, 11 43 605 1, 9, 17 1, 9, 17 49 470 VI 3, 11 3, 11 43 350 1, 9,17 1, 9, 17 48 832 7, 15 7, 15 14 917 VII 3, 11 3, 11 36 465 1, 9, 17 1,9, 17 42 712 7, 15 7, 15 11 857 8, 16 8, 16 45 772 VIII 3, 11 3, 11 34807 1, 9, 17 1, 9, 17 40 290 7, 15 7, 15 11 730 8, 16 8, 16 42 840 5, 135, 13 12 240

The relevant spots appear as a function of the nature of the analyteincubated, which shows that the hybridization reactions andimmunoreactions take place under the same operating conditions, inparticular in the same buffer and at the same temperature, even when thecapture partners are markers for different diseases.

1. A method for simultaneously detecting hybridization reactions andimmunoreactions in a sample which may contain target analytes consistingof at least one nucleic acid and of at least one other ligand that isdifferent in nature, characterized in that it comprises the stepsconsisting in: (i) depositing a known amount of volume of the samplediluted in a reaction buffer, onto a capture surface pre-coated with thepartners for capturing said target analytes, said capture partnersconsisting of at least one nucleic acid probe and at least oneantiligand, (ii) reacting at a temperature of between 15° C. and 60° C.and (iii) visualizing the hybridization reactions and immunoreactionsthus obtained.
 2. The method as claimed in claim 1, characterized inthat it comprises the following additional step (i′), placed betweenstep (i) and step (ii), consisting in: (i′) adding binding partnersspecific for at least one of said target analytes, which partners havebeen conjugated to labels beforehand.
 3. The method as claimed in claim1, characterized in that it comprises the following two additional steps(ii′) and (ii″), placed between step (ii) and step (iii), consisting in:(ii′) adding binding partners specific for at least one of said targetanalytes, which partners have been conjugated to labels beforehand, and(ii″) reacting at a temperature of between 15° C. and 60° C.
 4. Themethod as claimed in claim 2, characterized in that the target nucleicacid(s) contained in the biological sample has(have) been prelabeledsuch that the only binding partners to be added in step (i′) are thepartner(s) specific for the ligand, which partner(s) has(have) beenprelabeled.
 5. The method as claimed in claim 1, characterized in thatit comprises the following additional step (i′) placed between step (i)and step (ii), comprising: (i′) adding ligand(s) that has(have) beenprelabeled, which ligand(s) otherwise correspond to the ligand(s) thatmay be contained in the biological sample.
 6. The method as claimed inclaim 2, characterized in that the substrate(s) specific for thelabel(s) is(are) added before step (iii) for visualizing thehybridization reactions and immunoreactions.
 7. The method as claimed inclaim 1, characterized in that the capture surface is rinsed after steps(ii).
 8. The method as claimed in claim 1, characterized in that thereaction temperature is between 35 and 45° C.
 9. The method as claimedin claim 8, characterized in that the reaction temperature is between 37and 41° C.
 10. The method as claimed in claim 9, characterized in thatthe reaction temperature is 37° C. or 41° C.
 11. The method as claimedin claim 1, characterized in that the reaction buffer has an ionicstrength of between 0.4 and 1 M, has a pH of between 7 and 8, andcontains a surfactant.
 12. The method as claimed in claim 2,characterized in that the nucleic acid-specific binding partner(s) andthe ligand-specific binding partner(s) are labeled with the same label.13. The method as claimed in claim 1, characterized in that the capturepartners are markers for the same disease.
 14. The method as claimed inclaim 1, characterized in that the capture partners are markers fordifferent diseases.
 15. The method as claimed in claim 1, for detectingdiseases of infectious or metabolic origin.
 16. The method as claimed inclaim 15, for detecting diseases of viral origin.
 17. The method asclaimed in claim 1, for the industrial diagnosis of the presence ofbacteria.
 18. The method as claimed in claim 1, for identifying and/orquantifying biological molecules.
 19. The method as claimed in claim 1,for simultaneously detecting the transcriptome and the proteome of acell or of an organism.
 20. A diagnostic kit comprising a capturesurface, partners for capturing the target analytes consisting of atleast one nucleic acid probe and at least one other antiligand that isdifferent in nature, and a reaction buffer.
 21. The diagnostic kit asclaimed in claim 20, characterized in that it also comprises bindingpartners specific for the target ligands, that are prelabeled.
 22. Thediagnostic kit as claimed in claim 20, characterized in that it alsocomprises prelabeled ligands which may be contained in the sample to betested, and a reaction buffer.
 23. The diagnostic kit as claimed inclaim 21, characterized in that it also comprises binding partnersspecific for the target nucleic acids.
 24. The method as claimed inclaim 3, characterized in that the target nucleic acid(s) contained inthe biological sample has(have) been prelabeled such that the onlybinding partners to be added in step (ii′) are the partner(s) specificfor the ligand, which partner(s) has(have) been prelabeled.
 25. Themethod as claimed in claim 1, characterized in that it comprises thefollowing two additional steps (ii′) and (ii″), placed between step (ii)and step (iii), comprising: (ii′) adding ligand(s) that has(have) beenprelabeled, which ligand(s) otherwise corresponds to the ligand(s) thatmay be contained in the biological sample, and (ii″) reacting at atemperature of between 15° C. and 60° C.
 26. The method as claimed inclaim 5, characterized in that the target nucleic acid(s) contained inthe biological sample has(have) been prelabeled.
 27. The method asclaimed in claim 5, characterized in that, in step (i′), prelabeledbinding partner(s) specific for the target nucleic acid(s) are added.28. The method as claimed in claim 25, characterized in that the targetnucleic acid(s) contained in the biological sample has(have) beenprelabeled.
 29. The method as claimed in claim 25, characterized inthat, in step (ii′), prelabeled binding partner(s) specific for thetarget nucleic acid(s) are added.
 30. The method as claimed in claim 3,characterized in that the capture surface is rinsed after steps (ii)and/or (ii″).